Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field.


INTRODUCTION
Arachidonic acid (AA) is an essential ω-6 20 carbon polyunsaturated fatty acid that is abundant in the phospholipids of cellular membrane. In response to a stimulus, phospholipase A2 promotes its cleavage from the membrane and release into the cytosol, where it can be metabolized, leading to different classes of eicosanoids via three pathways ( Figure 1). 1, 2 The cyclooxygenase (COX) pathway catalyzes the production of prostaglandins, prostacyclins, and thromboxanes, endowed with inflammatory properties. The lipoxygenase (LOX) pathway generates leukotrienes, which play a significant part in the onset of asthma, arthritis, allergy, and inflammation. 3 Both pathways have been extensively studied and targeted pharmaceutically. 4−6 More recently, increasing attention is being paid to the third branch of the AA cascade, the cytochrome P450 (CYP) pathway that notably converts AA to epoxyeicosatrienoic acids (EETs). 7 EETs exhibit antihypertensive, anti-inflammatory, and anti-nociceptive properties, 8 but they are rapidly degraded by the soluble epoxide hydrolase (sEH, EPHX2, E.C. 3.3.2.10) to the less active or inactive dihydroxyeicosatrienoic acids (DHETs).
Therefore, sEH inhibition may lead to elevated levels of EETs thereby maintaining their beneficial properties. 9,10 Indeed, the use of selective sEH inhibitors (sEHI) in vivo models resulted in an increase of EETs levels and the reduction of blood pressure and inflammatory and pain states. Thus, sEH has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. 11−16 Given that sEH presents a hydrophobic pocket, several potent sEHI developed in the last years feature an adamantane moiety or an aromatic ring in their structure, such as AR9281, 1, and EC5026, 3, two of the sEHI that have reached clinical trials. 17,18 The first to enter was the adamantane-based AR9281, by Arete Therapeutics, for the treatment of hypertension in diabetic patients. However, it failed largely because of its poor pharmacokinetic properties but also poor target residence time on sEH and only moderate potency on the target. 17 Very recently, EicOsis has replaced the adamantane moiety of AR9281 by an aromatic ring for its drug candidate EC5026, currently in phase 1 clinical trials for the treatment of neuropathic pain. 18 Interestingly, both clinical candidates present similar structures: a left-hand side (lhs) hydrophobic moiety (black), a urea group (green), a piperidine residue (blue), and a right-hand side (rhs) acyl group (red). Also, EicOsis is currently advancing the analogue t-TUCB, 4, for veterinary clinical trials ( Figure 2). 19 Our recent observation that the lipophilic cavity of the enzyme is flexible enough to accommodate polycyclic units larger than adamantane, 20 led to the discovery of a new family of benzohomoadamantane-based ureas, such as 5 and 6, endowed with low nanomolar or even subnanomolar potencies ( Figure 2). 21 Further in vitro studies with these compounds demonstrated that while compound 5 presented moderate experimental solubility and very poor stability in human and mouse microsomes, compound 6 was endowed with favorable drug metabolism and pharmacokinetics (DMPK) properties and showed efficacy in an in vivo murine model of acute pancreatitis. 21 Later on, in an effort for improving the DMPK properties of piperidine 5, we designed a series of analogues where the urea core was replaced by an amide group. Although most of these amides retained or even improved the inhibitory activity of their urea counterparts at the human and mouse enzymes (e.g., compound 7, Figure 2), only moderate improvements in microsomal stabilities were found. 22 Herein, we report further medicinal chemistry around inhibitor 5. New piperidine derivatives retaining the urea group as the main pharmacophore, different substituents in the C-9 position of the polycyclic scaffold (R in I), and a broad selection of substituents at the nitrogen atom of the piperidine (R′ in I) were synthesized (Figure 2). After a screening cascade, two selected candidates with highly improved DMPK properties were subsequently studied in the murine model of capsaicin-induced allodynia. Finally, the best compound was evaluated in a murine model of visceral pain.
isocyanates II, followed by the addition of the required substituted aminopiperidine of general structure III to form the final ureas 9−25 (Scheme 1).
All the new compounds were fully characterized through their spectroscopic data and elemental analyses or highperformance liquid chromatography (HPLC)/mass spectrometry (MS) (see the Experimental Section and the Supporting Information for further details).

sEH Inhibition and Microsomal Stability.
Compound 5 presented high inhibitory activities against the human and murine enzymes and moderate experimental aqueous solubility (38 μM), but unacceptable stability in human and murine microsomes (Table 1). 21 Because the acyl chain of piperidine-based sEHI is known to be a suitable position for metabolism, 27 we decided to explore first new piperidine derivatives replacing the acetyl group of 5 by other fragments selected from previous other series of known sEHI to improve the microsomal stability. 28,29 Compounds 9−12 were synthesized maintaining the methyl group in the position R of the benzohomoadamantane scaffold I and replacing the acetyl group of 5 by the propionyl, tetrahydro-2H-pyran-4-carbonyl, isopropylsufonyl, and cyclopropanecarbonyl groups, respectively (Scheme 1). The inhibitory activity against the human and murine enzymes of the new ureas was evaluated, as well as their stabilities in human and mouse microsomes (Table 1).
Gratifyingly, regardless of the substituent of the piperidine ring, all the compounds showed potency in the low nanomolar or even subnanomolar ranges in both the human and murine enzymes (Table 1). Indeed, the most potent compound, 12, presented inhibitory activities in the subnanomolar range for both enzymes. However, except for 12, the microsomal stability of these new ureas was very poor and not improved from that of 5 (Table 1).
Consequently, we moved to another strategy for improving the microsomal stability of the compounds, by exploring the C-9 position of the benzohomoadamantane scaffold, replacing the methyl group in 5 and 9−12 by other substituents, such as halogen atoms or polar groups. The potency of these  compounds was measured against the human and murine enzymes ( Table 2). On the one hand, as expected considering that the catalytic center of sEH is highly hydrophobic, the compounds bearing a polar group in C-9, 23, and 24, presented higher IC 50 values than 5. Of note, the most important drop in the inhibitory activity was produced by the replacement of the methyl group of 5 by the polar hydroxyl group, compound 23. On the other hand, when the methyl group was replaced by chlorine or fluorine atoms, the inhibitory activities against the human and murine enzymes were maintained or even improved, as most of them presented IC 50 values in the low nanomolar or the subnanomolar range (Table 2).
Next, the microsomal stability of the most potent compounds was evaluated. Pleasingly, all the compounds featuring halogen atoms in the R position of the benzohomoadamantane scaffold presented better stabilities in human and mice microsomes than their methyl counterparts (Table 2). Especially, the chlorinated compounds 16, 18, and 19 exhibited excellent microsomal stabilities in the two species.

In Silico Study: Molecular Basis of Benzohomoadamantane/Piperidine-Based Ureas as sEH Inhibitors.
Next, the mechanism of binding of two compounds with high inhibitory activity, that is, 15 (R = Cl, R′ = tetrahydro-2Hpyran-4-carbonyl) and 21 (R = F, R′ = tetrahydro-2H-pyran-4carbonyl), was investigated with molecular dynamics (MD) simulations. sEHs present a flexible L-shaped active site pocket divided into three regions: the lhs and the rhs pockets that are connected by a central narrow channel defined by catalytic residues Asp335, Tyr383, and Tyr466 (see Figure 4). Recently, we showed that bulky benzohomoadamantane groups occupy the lhs in urea-based sEHIs that present both adamantyl and phenyl moieties, for example, compound 6. 21 However, available X-ray structures of sEH in complex with piperidinebased ureas show that the piperidine group can also occupy the lhs. 31 To determine the preferred binding mode of 15 and 21 that present both benzohomoadamantane and piperidine groups, we performed conventional MD simulations starting from two possible orientations in the sEH active site predicted  by molecular docking calculation (see the Experimental Section): (a) with the benzohomoadamantane in the lhs and piperidine in the rhs (see Figure 4a, similar to adamantyl based-urea in PDB 5AM3) and (b) the piperidine group is placed in lhs while benzohomoadamantane occupies rhs (similar to piperidine based-urea in PDB 5ALZ). 31 From these MD simulations, the binding affinity of 15 and 21 was estimated with molecular mechanics with generalized Born and surface area solvation (MM/GBSA) calculations showing that the orientation shown in Figure 4a is −5.7 and −10.2 kcal/mol more stable than the opposite orientation for compounds 15 and 21, respectively (see Table S2). When the benzohomoadamantane occupies the lhs and the piperidine the rhs, both compounds present similar absolute binding affinities (−68.0 and −69.4 kcal/mol for 15 and 21, respectively), which is in line with the similar IC 50 values. To corroborate these results, accelerated MD (aMD) simulations were performed to completely reconstruct the binding pathway of compound 15 into the sEH active site pocket (see Movie S1, Figure S1, and Experimental Section). This strategy is frequently used to predict substrate and inhibitor binding pathways in enzymes. 32,33 Spontaneous binding aMD simulations show how the inhibitor is recognized in the lhs pocket by the benzohomodamantane scaffold and then extends through the sEH binding site accommodating the benzohomoadamantane moiety in the lhs, while the piperidine counterpart lays in the  50 values are the average of three replicates. The fluorescent assay as performed here has a standard error between 10 and 20%, suggesting that differences of twofold or greater are significant. Because of limitations of the assay, it is difficult to distinguish among potencies <0.5 nM. 30 b Percentage of remaining compound after 60 min of incubation with pooled human and mouse microsomes in the presence of NADPH at 37°C. c ND: not determined. rhs pocket. Considering these results, we conclude that the orientation shown in Figure 4a is the preferred binding mode of compounds 15 and 21.
To understand in more detail the molecular basis of the inhibitory mechanism of benzohomoadamantane/piperidinebased ureas 15 and 21, the non-covalent interactions between the selected inhibitors and the active site residues of sEH were studied (see Figure 4 for compound 21 and Figure S2 for compound 15). MD simulations show that the inhibitor is retained in the active site through three strong hydrogen bond interactions between the urea moiety and the central channel residues Asp335, Tyr383, and Tyr466 (see Figures 4b and S3). In the rhs pocket, the piperidine group is stabilized through persistent hydrophobic interactions with His494 and Met419, while the tetrahydro-2H-pyran moiety is retained by the side chains of Leu417 and Trp525. The oxygen of tetrahydro-2H- Hydrogen bonds between the oxygens of the tetrahydropyran group of 21 and the hydrogen of the OH group of Ser415 is shown. The hydrophobic interaction average distances are computed between the terminal heavy atom of amino acid side chains and the centroid of each ring. Hydrogen bond distances between the carboxylic group of the catalytic Asp335 and the amide groups of the inhibitor and the distance between the carbonyl group of the urea inhibitor and the OH group of Tyr383 and Tyr466 residues. (c) Most relevant molecular interactions in the lhs. Average distances (in Å) obtained from the three replicas of 500 ns of MD simulations are represented. The CH−π interaction is calculated between the hydrogens of the benzohomoadamantane unit and the centroid of the benzoid ring of Trp336. The NH−π interaction is monitored between the amide hydrogen of Gln384 and the center of the aromatic ring of the benzohomoadamantane scaffold. pyran ring establishes transient hydrogen bonds with Ser415 and is relatively solvent exposed (see Figure 4b). In the lhs pocket, the orientation of the benzohomoadmantane moiety is directed by the NH···π interaction between the Gln384 and the aromatic ring of the polycyclic scaffold, which is maintained along the MD simulations. Additionally, hydrophobic interactions are established with the side chains of Met339 and Trp336. This extensive network of hydrophobic interactions and hydrogen bonds in the sEH pocket is key to recognize and bind the inhibitor in the active site.
Introducing a polar hydroxy group in the polycyclic scaffold (compound 23) significantly decreases the resulting inhibitory activity (see Table 2). To determine the molecular basis of this drop in activity, the binding modes of compounds 13 (R = Cl and R′ = acetyl and IC 50 = 1.6 nM) and 23 (R = OH and R′ = acetyl and IC 50 = 207 nM) were compared with MD simulations. The incorporation of OH in the polycyclic scaffold causes a series of rearrangements in the lhs pocket that destabilize the inhibitor bounds with the enzyme in the active site (see Figure S4). In particular, the Thr360 side chain establishes a hydrogen bond with the oxygen of the hydroxyl substituent of compound 23 that induces the rotation of the benzohomoadamantane scaffold in the lhs pocket. This breaks the NH−π interaction between Gln384 and the aromatic ring of 23 providing more flexibility to the benzohomadamantane moiety as compared to 13, 15, and 21, which may be related to the decreased activity (see Figure S5). In addition, the enhanced dynamism of the polycyclic scaffold allows the transient entrance of few water molecules into the lhs pocket (average number of water molecules 0.97 ± 0.96 for 23 and 0.31 ± 0.5 for 21, see Figure S6). Compound 24 (R = OCH 3 and R′ = acetyl, IC 50 = 48 nM) that also present reduced activity shows a similar behavior as 23 (see Figures S5 and S6). Therefore, the above-mentioned results and those previously reported with related compounds, 21 reveal that the presence of a small, lipophilic group at C-9 of the benzohomodamantane scaffold is key for the stability and activity of benzohomoadamantane-based sEHIs at the molecular level.
2.4. Further DMPK Profiling of the Selected Inhibitors. The halogen-substituted sEHI compounds that exhibited outstanding inhibitory activities and had more than 50% of the parent compound unaltered after incubation with human and/or murine microsomes were selected for further evaluation. Solubility, permeability through the blood−brain barrier (BBB), cytotoxicity, and cytochrome inhibition of the selected compounds 14−19, 21, 22, and 25 were experimentally measured. In addition, we evaluated all the synthesized compounds as pan assay interference compounds (PAINS) using SwissADME and FAFDrugs4 web tools. 34,35 None of them gave positive as PAINS.
While compounds 14, 16, 17, 18, and 19 exhibited limited solubility, with values lower than 20 μM, compounds 15, 21, 22, and 25 displayed good to excellent solubility values. Additionally, the selected compounds were further tested for predicted brain permeation in the widely used in vitro parallel artificial membrane permeability assay−BBB (PAMPA−BBB) model. 36 Compounds 14,15,22, and 25 showed CNS+ proving their potential capacity to reach CNS, whereas the other compounds presented uncertain BBB permeation (CNS +/−). Next, the cytotoxicity of the new sEHI was tested using the propidium iodide (PI) and MTT assays in SH-SY5Y cells. Interestingly, none of the selected compounds appeared cytotoxic at the highest concentration tested (100 μM) ( Table 3).
Finally, inhibition of several cytochrome P450 enzymes were measured, giving special attention to CYPs 2C19 and 2C9, as these isoforms are two of the main producers of EETs, the substrates of the sEH. 8 Unfortunately, compounds 16, 17, 18, and 19 inhibited significantly CYP 2C19. In contrast, compounds 14, 15, 21, 22, and 25 did not significantly inhibit these subfamilies of cytochromes (Table 3). Additionally, CYPs 2D6, 1A2, and 3A4 were also evaluated (Table S3). With the only exception of 25, which inhibited CYP3A4 in the submicromolar range, all the compounds showed IC 50 values higher than 10 μM (Tables 3 and S3).
After performing the above-mentioned screening cascade, three compounds, 15, 21, and 22, emerged as the more promising candidates. These compounds exhibited excellent inhibitory activities against the human and murine enzymes, improved metabolic stability, good solubility, and did not significantly inhibit cytochromes. Notwithstanding, hERG inhibition and Caco-2 assays were also performed in order to additionally characterize them. None of the compounds significantly inhibit hERG at 10 μM, and they displayed moderate permeability in Caco-2 cells. Finally, they were tested for selectivity against hCOX-2 and hLOX-5, two enzymes involved in the AA cascade. Gratifyingly, they did not present significant inhibition of these enzymes (Table 4).

sEH Engagement and Off-Target Profile.
Compound 28 was designed as a chemical probe with the objective to disturb the parent compound structure as little as possible. Important in this design was the knowledge that the piperidine nitrogen atom can be substituted without loss of biological activity. Therefore, a butynyl diazirinyl propionic acid minimalistic linker was coupled, via a straightforward amide coupling reaction, to the piperidine nitrogen of 27, in turn obtained from 8d through urea formation and Boc-removal (Scheme 2). The probe 28 was found to be a potent inhibitor with IC 50 of 0.5 and 0.4 nM, for the human and mouse enzymes, respectively.
Next, we tested whether probe 28 could covalently bind endogenously expressed human sEH in a complex proteome. Hence, photoaffinity labeling was followed by incorporation of an azide-TAMRA-Biotin tag via copper(I) azide alkyne cycloaddition (CuAAc). This tag allows both visualization and isolation of the probe's protein targets. A fluorescent band at 72 KDa was identified as sEH via immunoblotting (Figures 5, S7, S9 and S10).
Once the probe engagement of EPHX2 was confirmed, we determined the minimal probe labeling concentration using purified recombinant human EPHX2 ( Figure S8). The minimal probe concentration was found to be 100 nM, which was then used to get insights in the selectivity of the probe 28 and compound 15. Although it was observed that probe 28 labeled multiple bands, competition with the parent  compound 15 shows competition of only EPHX2, illustrating that this is the sole target with high occupancy and that the other bands are non-specific labeling events by the probe ( Figure 5). To further confirm the selective character of 15, we wanted to exclude p38 mitogen-activated protein kinase (p38 MAPK) and pro-angiogenic kinase vascular endothelial growth factor receptor-2 (VEGFR2) as targets because some ureabased sEHI are reported to show cross-reactivity with these proteins. 37−39 In addition, we also aimed to exclude membrane bound microsomal epoxide hydrolase as a possible off-target. 40 To this end, we performed pull-down experiments and immunoblotting with specific antibodies. These experiments confirmed that none of these proteins are targets of 28, underlining its selectivity ( Figure 5b).

Pharmacokinetic Study of Compounds 15 and 21.
Overall, compounds 15 and 21, with similar DMPK properties and structures, were selected for in vivo studies. First, a study was conducted in order to determine the pharmacokinetic profile in the plasma of compounds 15 and 21 when administered by a subcutaneous (sc) route at a single dose of 5 mg/kg. As shown in Table 5, absorption of 21 is fast, reaching C max (19.1 μg/mL) at 15 min after dosing. The compound disappeared from the plasma progressively and halflife (HL) was calculated to be around 0.7 h. In the case of 15, C max (1.2 μg/mL) was 15 times lower than that of 21, however, showing a higher HL (3.4 h). For both compounds, the narrow differences in AUC 0 t and AUC 0 ∞ showed complete exposure and good bioavailability. Although 21 demonstrated better bioavailability characteristics than 15 both compounds were subsequently evaluated in vivo efficacy studies.

In Vivo Efficacy Studies.
A first in vivo efficacy study was performed in a capsaicin-induced secondary mechanical hypersensitivity (allodynia) model in mice. It is well known that the increase in sensitivity to mechanical stimulation in the area surrounding capsaicin injection results from central sensitization, 41 which is a key process in chronic pain development and maintenance. 42 In our experimental conditions, mice markedly decreased their paw withdrawal latency to mechanical stimulation after capsaicin administration ( Figure 6), denoting the development of mechanical allodynia. The sc administration of the prototypic, brain-penetrant, 43−46 sEHI AS2586114 induced a dose-dependent reversion of the capsaicin-induced mechanical hypersensitivity reaching a full reversal of sensory hypersensitivity at 10 mg/kg ( Figure 6). The sc administration of compounds 15 and 21 fully inhibited mechanical hypersensitivity in a dose-dependent manner and with a much higher potency than AS2586114, reaching full reversal of sensory gain with 5 mg/kg for compound 15 and even with a dose as low as 1.25 mg/kg for compound 21 ( Figure 6), in spite of its limited predicted BBB permeability (as previously commented). Importantly, the administration of N-methanesulfonyl-6-(2-proparyloxyphenyl)hexanamide (MS-PPOH), an inhibitor of microsomal CYP450s, which is responsible for the production of EETs, 47 fully abolished the effect of not only AS2586114 but also those induced by compounds 15 and 21 ( Figure 6). These results strongly suggest that the three tested compounds induced the reversal of capsaicin-induced mechanical hypersensitivity through the in vivo inhibition of sEH.  Section and Tables S4 and S5 and Figures S12 and S13 in the Supporting Information. Given that the tested compounds induced ameliorative effects on this behavioral model of central sensitization attributable to sEH inhibition, we tested the effect of compound 21 (the most potent compound among the sEHI evaluated), in a model of pathological pain. Specifically, cyclophosphamide (CTX)-induced cystitis because it has been used as a model of interstitial cystitis/bladder pain syndrome, 48 and it is known that pain induced by this disease has a strong component of central sensitization in both humans and rodents. 49,50 In our experimental conditions, mice treated with CTX showed a significant increase in the pain behavioral score in comparison to mice treated with the vehicle (Figure 7a). The sc administration of compound 21 (0.63−2.5 mg/kg) significantly reduced this pain-related score in a dosedependent manner (Figure 7a). In addition, animals administered with the CTX vehicle showed a marked reduction in their mechanical threshold in the abdomen, denoting the development of referred hyperalgesia (Figure 7b). The sc treatment with compound 21 also reversed, in a dosedependent manner, the mechanical referred hyperalgesia induced by CTX ( Figure 7b). The administration of MS-PPOH fully reversed the effect of compound 21 in either the pain-related behaviors as in referred hyperalgesia (Figure 7a,b, respectively), mirroring the results obtained on capsaicininduced secondary hyperalgesia and suggesting that compound 21 exerted its in vivo effects on pain through sEH inhibition. To our knowledge, there are no previous studies exploring the role of sEHI on visceral pain. Therefore, our results suggest interstitial cystitis/pain bladder syndrome as a possible new indication for inhibitors of sEH.

CONCLUSIONS
sEH is a suitable target for several inflammatory and painrelated diseases. In this work, we report further medicinal chemistry around new benzohomoadamantane-based piperidine derivatives, analogues of the clinical candidates AR9281 and EC5026. The introduction of a halogen atom in the position C-9 of the benzohomoadamantane scaffold led to very potent compounds with improved DMPK properties. The in vitro profiling of these new sEHI (solubility, cytotoxicity, metabolic stability, CYP450s, hLOX-5, hCOX-2, and hERG inhibition) allowed one to select two suitable candidates for in vivo efficacy studies. The administration of compounds 15 and 21 reduced pain in the capsaicin-induced murine model of allodynia in a dose-dependent manner and outperformed AS2586114. Moreover, compound 21 was tested in a CTXinduced murine model of cystitis, revealing its robust analgesic effect. Hence, this study opens a whole range of applications of the benzohomoadamantane-based sEHIs in pain and likely other fields.

Chemical Synthesis.
Commercially available reagents and solvents were used without further purification unless stated otherwise. Preparative normal phase chromatography was performed on a CombiFlash Rf 150 (Teledyne Isco) with pre-packed RediSep Rf silica gel cartridges. Thin-layer chromatography was performed with aluminum-backed sheets with silica gel 60 F254 (Merck, ref 1.05554), and spots were visualized with UV light and 1% aqueous solution of KMnO 4 . HPLC purification was performed on a Prominence ultrafast liquid chromatography system (Shimadzu) using a Waters Xbridge 150 mm C18 prep column with a gradient of acetonitrile in water (with 0.1% trifluoroacetic acid) over 32 min. All compounds showed a sharp melting point and a single spot on TLC. Purity >95% of all final compounds was assessed by the integration of LC chromatograms. Melting points were determined in open capillary tubes with a MFB 595010M Gallenkamp. 400 MHz 1 H and 100.6 MHz 13 C NMR spectra were recorded on a Varian Mercury 400 or on a Bruker 400 Avance III spectrometers. 500 MHz 1 H NMR spectra were recorded on a Varian Inova 500 spectrometer. The chemical shifts are reported in ppm (δ scale) relative to internal tetramethylsilane, and coupling constants are reported in Hertz (Hz). Assignments given for the NMR spectra of selected new compounds have been carried out on the basis of DEPT, COSY 1H/ 1H (standard procedures), and COSY 1 H/ 13 C (gHSQC and gHMBC sequences) experiments. IR spectra were run on PerkinElmer Figure 7. Effects of compound 21 on pain-related behaviors and referred mechanical hyperalgesia induced by CTX. (a) Behavioral score was recorded at 30 min intervals over the 150−240 min observation period after the intraperitoneal (ip) injection of (CTX, 300 mg/kg) or its vehicle. (b) 50% mechanical threshold was evaluated by stimulation of the abdomen with von Frey filaments at 240 min after the administration CTX or its vehicle and was used as an index of referred hyperalgesia. Each bar and vertical line represents the mean ± SEM of values obtained in at least six animals per group. Statistically significant differences: **p < 0.01, between nonsensitized mice (open bar) and the other experimental groups; #p < 0.05, ##p < 0.01 between CTX-treated mice injected with the sEHI or their solvent (black bar); ++p < 0.01 mice injected with compound 21 associated or not with MS-PPOH (one-way ANOVA followed by Student−Newman−Keuls test). spectrum RX I, PerkinElmer spectrum TWO, or Nicolet Avatar 320 FT-IR spectrophotometers. Absorption values are expressed as wavenumbers (cm −1 ); only significant absorption bands are given. High-resolution mass spectrometry (HRMS) analyses were performed with an LC/MSD TOF Agilent Technologies spectrometer. The elemental analyses were carried out in a Flash 1112 series Thermo Finnigan elemental microanalyzer (A5) to determine C, H, N, and S. The structure of all new compounds was confirmed by elemental analysis and/or accurate mass measurement, IR, 1 H NMR, and 13 C NMR. The analytical samples of all the new compounds, which were subjected to pharmacological evaluation, possessed purity ≥95% as evidenced by their elemental analyses (Table S1) or HPLC/UV. HPLC/UV were determined with a HPLC Agilent 1260 Infinity II LC/MSD coupled to a photodiode array. 5 μL of sample 0.5 mg/mL in methanol/acetonitrile were injected, using an Agilent Poroshell 120 EC-C18, 2.7 μm, 50 mm × 4.6 mm column at 40°C. The mobile phase was a mixture of A = water with 0.05% formic acid and B = acetonitrile with 0.05% formic acid, with the method described as follows: flow 0.6 mL/min, 5% B−95% A 3 min, 100% B 4 min, and 95% B−5% A 1 min. Purity is given as % of absorbance at 220 nm.

annulen-7-yl)-3-(piperidin-4-yl)urea (27). t-
Each system was immersed in a pre-equilibrated truncated octahedral box of water molecules with an internal offset distance of 10 Å. All systems were neutralized with explicit counterions (Na + or Cl − ). A two-stage geometry optimization approach was performed. First, a short minimization of the positions of water molecules with positional restraints on the solute by a harmonic potential with a force constant of 500 kcal mol −1 Å −2 was done. The second stage was an unrestrained minimization of all the atoms in the simulation cell. Then, the systems were gently heated in six 50 ps steps, increasing the temperature by 50 K each step (0−300 K) under constant-volume, periodic-boundary conditions, and the particle-mesh Ewald approach 60 to introduce long-range electrostatic effects. For these steps, a 10 Å cutoff was applied to Lennard−Jones and electrostatic interactions. Bonds involving hydrogen were constrained with the SHAKE algorithm. 61 Harmonic restraints of 10 kcal mol −1 were applied to the solute, and the Langevin equilibration scheme was used to control and equalize the temperature. 62 The time step was kept at 2 fs during the heating stages, allowing potential inhomogeneities to self-adjust. Each system was then equilibrated for 2 ns with a 2 fs timestep at a constant pressure of 1 atm (NPT ensemble). Finally, conventional MD trajectories at a constant volume and temperature (300 K) were collected. In total, we carried out three replicas of 500 ns MD simulations for sEH in the presence of 13, 15, 21, 23, and 24 gathering a total of 7.5 μs of MD simulation time. Each MD simulation was clusterized based on active site residues, and the structures corresponding to the most populated clusters were used for the noncovalent interactions analysis. We monitored the presence of water molecules using the watershell function of the cpptraj MD analysis program. 63 aMD simulations 64,65 were used to study the spontaneous binding of 15 in the active site of sEH. Standard dualboost aMD simulations were performed using the same simulation protocols and aMD parameters as described in our previous works. 21 To reconstruct the spontaneous binding process, we placed one molecule of 15 in the solvent with a minimum distance of 25 Å from catalytic Asp335. First, we performed 250 ns of conventional MD followed by 10 replicas of 2 μs of aMD capturing one binding event (see Movie S1 comprising only the aMD simulation part). Binding affinities (kcal/mol) of compounds 13, 15, 21, and 23 were computed using the MM/GBSA method as implemented in AMBER 18.

Preparation of HEK 293T
Lysates. HEK293T cells were grown in DMEM media (D6546-500ML Sigma) supplemented with 10% FBS, 2 mM glutamax, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. They were maintained at 37°C with 5% CO 2 . Cells were split every 3 to 4 days according to an ATCC protocol. The cells were harvested and collected by centrifugation (500 g for 5 min at 4°C ) and the supernatant was removed. The pellets were washed twice with ice-cold PBS and resuspended in 2 vol of ice-cold lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT and 0.5% NP-40). After 30 min on ice, the cells were centrifuged to remove cell debris for 5 min at 4°C. The supernatant was aliquoted and flash frozen in liquid N 2 for use as lysates, with a total protein concentration of 1 mg/mL. Protein concentrations were determined using the BCA assay (Fisher Scientific).

Labeling in HEK 293T
Lysates. HEK 293T lysates were spiked or not with 100 ng of recombinant purified sEH, treated either with 100 nM probe 28 or DMSO, and incubated for 30 min at 37°C. After this time, the samples were irradiated for 6 min at 365 nm using a 100 W UV lamp. Subsequently, a bi-functional tag containing a TAMRA dye and a biotin was incorporated using copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC). The photoaffinity labeling was analyzed by in-gel analysis, mixing the samples with 4× SDS-loading buffer, and separating using 12% SDS-PAGE after which the gel was scanned on a Typhoon FLA 9500. 4.6. Labeling Purified Soluble Epoxide Hydrolase for Minimal Probe Concentration Determination. Purified recombinant sEH was produced and purified as indicated previously. 30 Of the pure active enzyme 100 or 200 ng were incubated for 30 min at 37°C with decreasing concentrations of probe 3, namely: 10 μM, 1 μM, 100 nM, 10 nM, and 1 nM. After this time, the compounds were irradiated for 6 min at 365 nm using a 100 W UV lamp. Subsequently, a bifunctional tag containing a TAMRA dye and a biotin was incorporated using copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC). The photoaffinity labeling was analyzed by in-gel analysis by mixing the samples with 4× SDS-loading buffer and separating using 12% SDS-PAGE after which the gel was scanned on a Typhoon FLA 9500. 4.7. EPHX2 Target Engagement Confirmation and Off-Target Elucidation by Pull Down. Untreated HEK293T whole cell lysates were normalized to a concentration of 1 mg/mL in a volume of 100 μL, per condition. Lysates were then treated with DMSO, 10 μM of probe 28 or 10 μM of probe 28, and 100 μM of 15 (for competition experiments), and incubated at 37°C for 30 min. After this time, the whole was irradiated for 6 min at 365 nm using a 100 W UV lamp. Subsequently, a bi-functional tag containing a TAMRA dye and a biotin was incorporated via CuAAC. The excess reagents from the samples were then removed by acetone precipitation. Following resuspension of the pellets to a final volume of 100 μL, half of the sample was kept as the input control. The remaining 50 μL were incubated with 20 μL of pre-washed streptavidin beads (Thermo Fisher) for 1 h with mixing at RT. The supernatant was removed, and the beads were sequentially washed with 0.33% SDS in PBS (2 × 50 μL), 1 M NaCl (2 × 50 μL) and PBS (2 × 50 μL). Bound proteins were eluted by boiling (95°C) the beads with 60 μL of 1× SDS loading buffer for 10 min. Samples were resolved by 12% SDS-PAGE. Following visualization using a Typhoon FLA 9500, the gel was transferred onto a nitrocellulose membrane and probed with VEGF2 (cell signaling), p38 MAPK (cell signaling), EPHX1 (Elabscience), and EPHX2 (Abcam) for detection.
This experiment was also carried out using lower probe and parent compound concentrations of 1 and 10 μM, respectively, yielding the same results.

Affinity-Based Probe and Parent Compound Off-Target Profile Elucidation.
To HEK293T cell lysates at 1 mg/mL protein concentration spiked or not with 100 ng of recombinant human sEH and 100 ng of purified recombinant enzyme were treated with either 100 nM probe 28, 10 μM urea 15, and 100 nM probe 28 or DMSO to a concentration of 1% of the total sample. After 30 min of incubation of the compounds at 37°C, the whole was irradiated for 6 min at 365 nm using a 100 W UV lamp. Subsequently, a bi-functional tag containing a TAMRA dye and a biotin was incorporated via CuAAC. The samples were analyzed by in-gel analysis by mixing the samples with 4× SDS-loading buffer and separating using 12% SDS-PAGE after which the gel was scanned on a Typhoon FLA 9500 and/or submitted to Western blot analysis using human sEH antibody for detection (Abcam). The comparison of labeling patterns via fluorescence showed the inability of the parent compound to compete out the probe 28 for most of the targets, which pointed out that except for the sEH the other labeled proteins are not targets of the parent compound but of the probe 28. The plasma was separated by centrifugation for 10 min and stored at −80°C until analysis by HPLC. Frozen plasma samples were thawed at room temperature and 25 μL of acetonitrile were added to a 100 μL of plasma sample. The sample was vortexed for 30 s and centrifuged (14,000 rpm/min) for 5 min. The supernatant was transferred to an injection bottle and 25 μL was injected into the chromatographic system.

Instruments and Analysis
Conditions. The HPLC system was a PerkinElmer LC (PerkinElmer INC, Massachusetts, U.S.) consisting of a Flexar LC pump, a chromatography interface (NCI 900 network), a Flexar LC autosampler PE, and a Waters 2487 dual λ absorbance detector. The chromatographic column was a kromasil 100-5-C18 (4,0 × 200 mm-Teknokroma Analitica S.A. Sant Cugat, Spain). Flow was 0.8 mL/min and the mobile phase consisting in 0.05 M KH 2 PO4 (30%)/acetonitrile (70%) in isocratic conditions. The elution times of 15 and 21 were 5.6 and 4.4 min, respectively. Compounds were detected at 220 nm. The assay had a range of 0.015−25 μg mL −1 . The calibration curves were constructed by plotting the peak area ratio of analyzed peak against known concentrations. Compound 22 was analyzed under the same chromatographic conditions but the response to the analysis was 10 times lower than that of 15 and 21.
4.9.4. Pharmacokinetic Analysis. 15 and 51 plasma concentrations versus time curves for the means of animals were analyzed by a noncompartmental model based on the statistical moment theory using the "PK Solutions" computer program. The pharmacokinetic parameters calculated were as follows: area under the plot of plasma concentration versus time curve (AUC), calculated using the trapezoidal rule in the interval 0−6 h; HL (t 1/2β ), determined as ln 2/β , being β, calculated from the slope of the linear, least-squares regression line; C max and T max were read directly from the mean concentration curves. 4.10. In Vivo Efficacy Studies. 4.10.1. Experimental Animals. Experiments were performed in female WT-CD1 (Charles River, Barcelona, Spain) mice weighing 25−30 g. Mice were acclimated in our animal facilities for at least 1 week before testing and were housed in a room under controlled environmental conditions: 12/12 h day/ night cycle, constant temperature (22 ± 2°C), air replacement every 20 min, and they were fed a standard laboratory diet (Harlan Teklad Research Diet, Madison, WI, USA) and tap water ad libitum until the beginning of the experiments. The behavioral test was conducted during the light phase (from 9.00 to 15.00 h), and randomly throughout the oestrous cycle. Animal care was in accordance with institutional (Research Ethics Committee of the University of Granada, Spain), regional (Junta de Andalucía, Spain), and international standards (European Communities Council Directive 2010/ 63).

Drugs and Drug
Administration. The sEHI were dissolved in 5% DMSO (Merck KGaA, Darmstadt, Germany) in physiological sterile saline (0.9% NaCl). Drug solutions were prepared immediately before the start of the experiments and injected sc in a volume of 5 mL/kg into the interscapular area. To test for the effects of MS-PPOH (Cayman Chemical Company, Ann Arbor, MI, USA), a selective inhibitor of microsomal CYP450 epoxidase, 45 on the effects induced by the sEHI tested, this compound was dissolved in DMSO 5% and cyclodextrin 40% in saline and administered 5 min before sEHI injection. When the effect of the association of several drugs was assessed, each injection was performed in different areas of the interscapular zone to avoid the mixture of the drug solutions and any physicochemical interaction between them. In all cases, the researchers who performed the experiments were blinded to the treatment received by each animal.
As it will be detailed below, we used two different algogenic substances to explore the effects of sEHI on nociception: capsaicin was used to induce somatic mechanical hypersensitivity, and CTX to induce visceral pain. Capsaicin (Sigma-Aldrich Química S.A.) was dissolved in 1% DMSO in physiological sterile saline to a concentration of 0.05 μg/μL (i.e., 1 μg per mouse). Capsaicin solution was injected intraplantarly (i.pl.) into the right hind paw proximate to the heel, in a volume of 20 μL using a 1710 TLL Hamilton microsyringe (Teknokroma, Barcelona, Spain) with a 30 1/2gauge needle. Control animals were injected with the same volume of the vehicle of capsaicin. CTX (Sigma-Aldrich, Madrid, Spain), which was used to induce a painful cystitis, was dissolved in saline and injected ip at a dose of 300 mg/kg, in a volume of 10 ml/kg. The same volume of solvents was injected in control animals.

Evaluation of Capsaicin-Induced Secondary Mechanical
Hypersensitivity. Animals were placed into individual test compartments for 2 h before the test to habituate them to the test conditions. The test compartments had black walls and were situated on an elevated mesh-bottomed platform with a 0.5 cm 2 grid to provide access to the ventral surface of the hind paws. In all experiments, punctate mechanical stimulation was applied with a dynamic plantar aesthesiometer (Ugo Basile, Varese, Italy) at 15 min after the administration of capsaicin or its solvent. Briefly, a nonflexible filament (0.5 mm diameter) was electronically driven into the ventral side of the paw previously injected with capsaicin or solvent (i.e., the right hind paw), at least 5 mm away from the site of the injection toward the fingers. The intensity of the stimulation was fixed at 0.5 g force, as described previously. 66 When a paw withdrawal response occurred, the stimulus was automatically terminated, and the response latency time was automatically recorded. The filament was applied three times, separated by intervals of 0.5 min, and the mean value of the three trials was considered the withdrawal latency time of the animal. A cutoff time of 50 s was used. The compounds tested, or their solvent, were administered sc 30 min before the i.pl. administration of capsaicin or DMSO 1% (i.e., 45 min before we evaluated the response to the mechanical punctate stimulus).

Evaluation of Cyclophosphamide-Induced Visceral
Pain. CTX-evoked pain behaviors and referred hyperalgesia were examined following a previously described protocol with slight modifications. 48 Animals were placed into the same individual test compartments described above for 40 min to habituate them to the test conditions. Then, mice were injected ip with CTX or saline. Compound 21 or its solvent was sc injected at 120 min after CTX ip administration, and pain behaviors were recorded for 2 min every 30 min in the period from 150 to 240 min. These pain-related behaviors were coded according to the following scale: 0 = normal, 1 = piloerection, 2 = labored breathing, 3 = licking of the abdomen, and 4 = stretching and contractions of the abdomen. At the end of the 2 h observation period (i.e., 4 h after the CTX injection), the sensory threshold in the abdomen was measured 240 min after CTX administration, using a series of von Frey filaments with bending forces ranging from 0.02 to 2 g (Stoelting, Wood Dale, USA). Testing was always initiated with the 0.4 g filament. The response to the filament was considered positive if immediate licking/scratching of the application site, sharp retraction of the abdomen, or jumping was observed. If there was a positive response, a weaker filament was used; if there was no response, a stronger stimulus was then selected. The 50% threshold withdrawal was determined using the up and down methods and calculated using the Up−Down Reader software. 67 ■ ASSOCIATED CONTENT
Complete details of in vitro biological methods, 1 H and 13